Frank-Kamenetsky M, Zhang XM, Bottega S, Guicherit O, Wichterle H, Dudek H et al.. Small-molecule modulators of Hedgehog signaling: identification and characterization of Smoothened agonists and antagonists. J Biol 1: 10

Curis, Inc., 61 Moulton Street, Cambridge, MA 02138, USA.
Journal of Biology 12/2002; 1(2):10. DOI: 10.1186/1475-4924-1-10
Source: PubMed

ABSTRACT The Hedgehog (Hh) signaling pathway is vital to animal development as it mediates the differentiation of multiple cell types during embryogenesis. In adults, Hh signaling can be activated to facilitate tissue maintenance and repair. Moreover, stimulation of the Hh pathway has shown therapeutic efficacy in models of neuropathy. The underlying mechanisms of Hh signal transduction remain obscure, however: little is known about the communication between the pathway suppressor Patched (Ptc), a multipass transmembrane protein that directly binds Hh, and the pathway activator Smoothened (Smo), a protein that is related to G-protein-coupled receptors and is capable of constitutive activation in the absence of Ptc.
We have identified and characterized a synthetic non-peptidyl small molecule, Hh-Ag, that acts as an agonist of the Hh pathway. This Hh agonist promotes cell-type-specific proliferation and concentration-dependent differentiation in vitro, while in utero it rescues aspects of the Hh-signaling defect in Sonic hedgehog-null, but not Smo-null, mouse embryos. Biochemical studies with Hh-Ag, the Hh-signaling antagonist cyclopamine, and a novel Hh-signaling inhibitor Cur61414, reveal that the action of all these compounds is independent of Hh-protein ligand and of the Hh receptor Ptc, as each binds directly to Smo.
Smo can have its activity modulated directly by synthetic small molecules. These studies raise the possibility that Hh signaling may be regulated by endogenous small molecules in vivo and provide potent compounds with which to test the therapeutic value of activating the Hh-signaling pathway in the treatment of traumatic and chronic degenerative conditions.

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Available from: Hynek Wichterle, Aug 22, 2014
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    • "The interaction of GCs with the Hh pathway leads to several important observations: First, all small molecules that induce ligand-independent Smo accumulation to the PC characterized to date either activate or inhibit Smo activity. Agonists include SAG and purmorphamine (Chen, et al., 2002; Frank-Kamenetsky, et al., 2002; Sinha and Chen, 2006). Cyc though an antagonist also induces Smo transolcation to the PC(Rohatgi, et al., 2009; Wang, et al., 2009; Wilson, et al., 2009). "
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    ABSTRACT: The Hedgehog signaling pathway is linked to a variety of diseases, notably a range of cancers. The first generation of drug screens identified Smoothened (Smo), a membrane protein essential for signaling, as an attractive drug target. Smo localizes to the primary cilium upon pathway activation, and this transition is critical for the response to Hedgehog ligands. In a high content screen directly monitoring Smo distribution in Hedgehog-responsive cells, we identified different glucocorticoids as specific modulators of Smo ciliary accumulation. One class promoted Smo accumulation, conferring cellular hypersensitivity to Hedgehog stimulation. In contrast, a second class inhibited Smo ciliary localization and signaling activity by both wild-type Smo, and mutant forms of Smo, SmoM2, and SmoD473H, that are refractory to previously identified Smo antagonists. These findings point to the potential for developing glucocorticoid-based pharmacological modulation of Smo signaling to treat mutated drug-resistant forms of Smo, an emerging problem in long-term cancer therapy. They also raise a concern about potential crosstalk of glucocorticoid drugs in the Hedgehog pathway, if therapeutic administration exceeds levels associated with on-target transcriptional mechanisms of glucocorticoid action.
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    • "As at E15.5 we found the highest levels of Gli1 expression adjacent to acTUBB3-positive cells in both wild type and orJ retinas (data not shown), which is consistent with the idea that signaling is most robust near the cells that produce the ligand. Second, we performed explant cultures of P0 orJ retinas in the presence or absence of cyclopamine, a pharmacological inhibitor of Hh signaling (Chen et al., 2002; Cooper et al., 1998; Frank-Kamenetsky et al., 2002; Incardona et al., 1998; Taipale et al., 2000). After a 16 hr culture period, we found Gli1 expression was reduced in response to cyclopamine treatment (Fig. 11A,C), while Vsx2 expression remained unchanged (Fig. 11B,D). "
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    ABSTRACT: Vertebrate retinal progenitor cells (RPCs) undergo a robust proliferative expansion to produce enough cells for the retina to form appropriately. Vsx2 (formerly Chx10), a homeodomain protein expressed in RPCs, is required for sufficient proliferation to occur. Sonic Hedgehog protein (SHH), secreted by retinal ganglion cells (RGCs), activates Hedgehog (Hh) signaling in RPCs and is also required for sufficient proliferation to occur. Therefore, we sought to determine if reduced Hh signaling is a contributing factor to the proliferation changes that occur in the absence of Vsx2. To do this, we examined Shh expression and Hh signaling activity in the homozygous ocular retardation J (orJ) mouse, which harbors a recessive null allele in the Vsx2 gene. We found that Shh expression and Hh signaling activity are delayed during early retinal development in orJ mice and this correlates with a delay in the onset of RGC differentiation. At birth, reduced expression of genes regulated by Hh signaling was observed despite the production of SHH ligand. orJ RPCs respond to pre-processed recombinant SHH ligand (SHH-N) in explant culture as evidenced by increased proliferation and expression of Hh target genes. Interestingly, proliferation in the orJ retina is further inhibited by cyclopamine, an antagonist of Hh signaling. Our results suggest that reduced Hh signaling contributes to the reduced level of RPC proliferation in the orJ retina, thereby revealing a role for Vsx2 in mediating mitogen signaling.
    Developmental Biology 06/2008; 317(2):560-75. DOI:10.1016/j.ydbio.2008.02.055 · 3.64 Impact Factor
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    • "To further confirm the ability to modulate Hh signaling in GS, we sought to establish whether, once activated, the pathway could then be inhibited. For this purpose, additional GS from a GII astrocytoma and a GIII anaplastic oligodendroglioma were generated in the presence or absence of a Smoothened (SMOH) agonist (SAG; see Figure 1) (Chen et al., 2002b; Frank-Kamenetsky et al., 2002). After 7 days, GS from the corresponding parent culture were re-plated either alone in the presence of SANT1 (a SMOH antagonist; see Figure 1) (Chen et al., 2002a) or in increasing doses of SAG. "
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    ABSTRACT: The hedgehog (Hh) signaling pathway regulates progenitor cells during embryogenesis and tumorigenesis in multiple organ systems. We have investigated the activity of this pathway in adult gliomas, and demonstrate that the Hh pathway is operational and activated within grade II and III gliomas, but not grade IV de novo glioblastoma multiforme. Furthermore, our studies reveal that pathway activity and responsiveness is confined to progenitor cells within these tumors. Additionally, we demonstrate that Hh signaling in glioma progenitor cells is ligand-dependent and provide evidence documenting the in vivo source of Sonic hedgehog protein. These findings suggest a regulatory role for the Hh pathway in progenitor cells within grade II and III gliomas, and the potential clinical utility of monitoring and targeting this pathway in these primary brain tumors.
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